Mutation Screening of Mtdna Combined Targeted Exon Sequencing in a Cohort with Suspected Hereditary Optic Neuropathy

Article Mutation Screening of mtDNA Combined Targeted Exon Sequencing in a Cohort With Suspected Hereditary Optic Neuropathy

Jian-Kang Li1–3,*,WeiLi1,3,*, Feng-Juan Gao5,6,*, Shou-Fang Qu7, Fang-Yuan Hu5,6, Sheng-Hai Zhang5,6, Li-Li Li7,Zi-WeiWang1,3,YongQiu3,4,Lu-ShengWang2,3, Jie Huang7, Ji-Hong Wu5,6, and Fang Chen3,4

1 BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China 2 Department of Computer Science, City University of Hong Kong, Kowloon, Hong Kong 3 BGI-Shenzhen, Shenzhen, China 4 MGI, BGI-Shenzhen, Shenzhen, China 5 Eye Institute, Eye, Ear, Nose and Throat Hospital, College of Medicine, Fudan University, Shanghai, China 6 Shanghai Key Laboratory of Visual Impairment and Restoration, Science and Technology Commission of Shanghai Municipality, Shanghai, China 7 National Institutes for Food and Drug Control, Tiantan Xili Dongcheng District, Beijing, China

Correspondence: Fang Chen, Purpose: Leber hereditary optic neuropathy (LHON) and autosomal dominant optic BGI-Shenzhen, Shenzhen 518083, atrophy (ADOA) are the two commonest forms of hereditary optic neuropathy. The China. e-mail: aim of this study was to comprehensively investigate the incidence and spectrum [email protected] of mutations in patients with suspected hereditary optic neuropathy by combining Ji-Hong Wu, Eye Institute, Eye and mitochondrial DNA (mtDNA) genome-wide and targeted exon sequencing. ENT Hospital, College of Medicine, Methods: Fudan University, Shanghai, China. A cohort of 1101 subjects were recruited to participate in the study, compris- e-mail: [email protected] ing 177 families (177 probands and their family members, a total of 537 subjects, includ- Jie Huang, National Institutes for ing 254 patients) and 164 sporadic cases with suspected hereditary optic neuropa- Food and Drug Control, No. 2, thy, and 400 unrelated control subjects for genetic analysis: all subjects (includ- Tiantan Xili Dongcheng District, ing control subjects) underwent a comprehensive ophthalmologic examination and Beijing 100050, China. e-mail: were subjected to sequencing analysis of mtDNA genome-wide and targeted exon. [email protected] Overall, targeted exon sequencing was used to screen 792 genes associated with common hereditary eye diseases, and the mtDNA genome-wide were screened by next- Received: December 5, 2019 generation sequencing. Accepted: April 23, 2020 Published: July 8, 2020 Results: We found variants detected in 168 (40.2%, 168/418) of the 418 patients screened. Among these, 132 cases (78.6%, 132/168) were detected with known LHON Keywords: Leber hereditary optic disease-causing mtDNA variants; 40 cases (23.8%, 40/168) were detected with nuclear neuropathy; autosomal dominant DNA (ntDNA) variants, which included 36 cases (21.4%, 36/168) with detected OPA1 optic atrophy; autosomal recessive mutations, 4 patients (2.4%, 4/168) with detected OPA3 mutations, and 2 patients optic atrophy; mtDNA; ntDNA; (1.2%, 2/168) with detected TMEM126A homozygous mutation. Coexistence variation panel-based targeted exon (mtDNA/mtDNA [n = 16], ntDNA/ntDNA [n = 4], mtDNA/ntDNA [n = 7]) was found sequencing in 27 patients (16.4%, 27/165), including mtDNA/ntDNA coexistence variation that was Citation: Li J-K, Li W, Gao F-J, Qu S-F, detected in seven patients. Among these ntDNA mutations, 38 distinct disease-causing Hu F-Y, Zhang S-H, Li L-L, Wang Z-W, variants, including autosomal recessive heterozygous mutations, were detected, which QiuY,WangL-S,HuangJ,WuJ-H, included 22 novel variants and two de novo variants. Total haplogroup distribution Chen F. Mutation screening of showed that 34.5% (29/84) and 28.6% (24/84) of the affected subjects with m.11778G>A mtDNA combined targeted exon belonged to haplogroup D and M, with a high frequency of subhaplogroups D4, D5, and sequencing in a cohort with M7. suspected hereditary optic Conclusions: The LHON-mtDNA mutations are the commonest genetic defects in this neuropathy. Trans Vis Sci Tech. OPA1 2020;9(8):11, Chinese cohort, followed by the mutations. To our knowledge, this is the first https://doi.org/10.1167/tvst.9.8.11 comprehensive study of LHON, ADOA, and autosomal recessive optic atrophy combined with mtDNA genome-wide and targeted exon sequencing, as well as haplogroup

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 2

analysis, in a large cohort of Chinese patients with suspected hereditary optic neuropathy. Our findings provide a powerful basis for genetic counseling in patients with suspected hereditary optic neuropathy. Translational Relevance: We applied mtDNA genome-wide sequencing combined with panel-based targeted exon sequencing to explore the pathogenic variation spectrum and genetic characteristics of patients with suspected hereditary optic neuropathy, providing a comprehensive research strategy for clinical assistant diagnosis, treatment, and genetic counseling.

Introduction geneous (mixed wild-type and mutant) or homogenous (mutant-only) forms. ADOA is a disorder characterized by degen- Hereditary optic neuropathy is a group of hered- erative optic nerve fibers, mainly involving RGCs itary heterogeneous diseases involving function and axons of optic nerve formation. The estimated change in the optic nerve and retinal ganglion cells 1 prevalence of the disease ranges from 1:12000 to (RGC), which causes variable vision decline. There 1:50000, characterized by early-onset loss of bilat- are several modes of inheritance for these disor- eral vision, defects in color vision, and typical tempo- ders, including Leber hereditary optic neuropathy ral pale optic nerve.15 Although several genes have (LHON, OMIM535000) and autosomal dominant been reported to be associated with ADOA, most optic atrophy (ADOA, OMIM165500), which are the studies are still associated with genetic mutations two commonest forms of these disorders caused by in OPA1 (OMIM605290) and OPA3 (OMIM606580) mutations in mitochondrial DNA (mtDNA) and 2–4 gene encoding mitochondrial proteins, most of which nuclear DNA (ntDNA). Autosomal recessive are anchored to the mitochondrial inner membrane optic atrophy (AROA) is a rare mode of inheri- and are ubiquitously expressed.16–18 Previous studies tance, and several reports of this recessive form of patients with ADOA have shown that most are have been linked to TMEM126A (OMIM612988), caused by mutations in the OPA1 gene, accounting for RTN4IP1 (OMIM610502), ACO2 (OMIM100850), 5–7,22 approximately 60% to 80%. The OPA1 gene encodes and YME1L1 (OMIM617302) genes. mitochondrial proteins located in the mitochondrial LHON is the commonest type of maternal hered- inner membrane and regulates the stability of the itary disorder caused by structural and functional mitochondrial network, mitochondrial bioenergetic abnormalities of mtDNA, with an estimated preva- 8,9 output, and isolation of proapoptotic cytochrome c lence of 1:50000 worldwide. It was first described oxidase molecules in the mitochondrial cristae spaces, by Theodor Leber as a maternally hereditary disor- and more than 300 mutations have been detected in the der of optic nerve degeneration that is usually occur- previous reports.15,19–21 ring in patients between the age of 18 and 35, with AROA is a rare disease that affects RGCs and the acute or subacute visual loss of the central region 10,11 nervous system. To date, it has been reported that this and preferentially affects men. LHON is a hetero- form of AROA is related to 4 causative genes, including geneous disorder caused by mtDNA mutations. The 3 variants detected in the transmembrane protein 126A composition of mtDNA includes 13 protein-coding gene (TMEM126A, OMIM612988) in 8 families, 14 genes, 2 ribosomal RNAs, and 22 tRNAs. Over variants detected in the reticulon 4-interacting protein 90% of LHON is caused by one of three primary 1gene(RTN4IP1, OMIM610502) in 17 families, 7 disease-causing variants, m.3460G>A, m.11778G>A, > variants detected in the aconitase 2 gene (ACO2, and m.14484T C, which involve the MT-ND1, MT- OMIM100850), and 1 variant detected in the YME1 ND4,andMT-ND6 gene encoding three subunits 8,11,12 Like 1 ATPase gene (YME1L1, OMIM617302) in of the respiratory chain complex I, respectively. 1family.5–7,22–24 There are overlapping clinical and Secondary mutations (such as MT-ND1 m.3394T>C > pathological features in almost all affected patients, in and MT-ND4 m.11696G A) often interact with which RGCs are prone to mitochondrial dysfunction.25 primary mutations to affect the phenotypic expression Among these causative genes, the TMEM126A gene of LHON, which is present in the LHON family, but at has been reported to be specifically associated with the same time appears in the control subjects at a lower 13,14 nonsyndromic AROA. The TMEM126A gene spans frequency than LHON patients. These mtDNA a genomic region of 8.5kb and consists of five exons, mutations associated with LHON occurred in hetero- and it has been mapped to chromosome 11q14.1-q21

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 3 within a critical interval associated with optic atrophy best-corrected visual acuity (BCVA)measurement, slit- identified by homozygosity mapping. In contrast to the lamp biomicroscopy examination, intraocular pressure OPA1 (OMIM605290) mutations, Hanein et al.5 found detection (IOP, Goldmann tonometry; Haag-Streit, no fragmentation of the mitochondrial network and/or UK), full-field electroretinography, fundus photo- depletion of the mtDNA in patient fibroblasts caused graph (Canon CR6-45NM fundus camera; Canon, by the TMEM26A mutation, which indicates that there Tokyo, Japan), and swept-domain optical coherence may be no functional correlation between TMEM26A tomography (Heidelberg Engineering Inc., Heidelberg, and OPA1. Thus far, three variants of the TMEM26A Germany). In addition, information associated with gene have been associated with AROA, including a the disease, including family history, age of onset, and recurrent R55X homozygous nonsense mutation that decreased subjective visual acuity, were collected. The has been detected in multiple families. Among these diagnosis of suspected hereditary optic neuropathy was families includes four families with autosomal recessive assessed by professional neuroophthalmologists. nonsyndromic optic atrophy and one family with optic atrophy combined auditory neuropathy, which sugges- tion that auditory neuropathy might be a key feature of Targeted Exon Sequencing and TMEM126A associated optic neuropathy.5,26 Next-Generation Sequencing Despite several previous studies that have described the genetic and clinical features of hereditary optic Peripheral blood samples from all participants were neuropathy, the overall status of LHON, ADOA, and collected at the Eye and ENT Hospital of Fudan AROA mutations remains to be determined in Chinese University. The mtDNA and ntDNA were extracted patients with suspected hereditary optic neuropathy. from whole blood using the MGIEasy Mitochon- In this study, we report the results of a comprehen- drial Whole Genome Amplification Kit (MGI, Inc., sive molecular analysis of 418 patients with suspected Shenzhen, China) and FlexiGene DNA Kit (Qiagen, hereditary optic neuropathy and detected molecu- Venlo, The Netherlands) according to the manufac- lar variability in nuclear and mitochondrial genomes turer’s protocols, respectively. In this study, customized based on next-generation sequencing. panel-based next-generation sequencing (NGS) and mtDNA genome-wide sequencing were performed for all participants. We collaborated with MGI- Materials and Methods Shenzhen (Shenzhen, Guangdong, China) to design the Target_Eye_792_V2 chip, which has exon capture and untranslated regions of 792 genes associated Subjects and Ethics Statement with common hereditary eye diseases (Supplementary 27,28 A cohort of 1101 subjects, including 177 families Table S1). On average, the mean coverage depth (177 probands and their relatives) and 164 sporadic was more than 300X for ntDNA, the mean cover- subjects with suspected hereditary optic neuropathy age depth was more than 30X for mtDNA, and the (total patients: 418; total probands: 341) and 400 coverage of target region was close to 99.9% using unrelated control subjects were recruited at the Eye the MGISEQ-2000 (DNBSEQ-G400) platform (MGI, and Ear, Nose, and Throat (ENT) Hospital of Fudan Inc., Shenzhen, China). University from February 2016 to June 2019. The study was permitted by the ethics committee of the Eye and ENT Hospital of Fudan University and adhered to Bioinformatics Analysis the tenets of the Declaration of Helsinki. Individuals The target exon sequencing reads are aligned to participating in the study signed informed consent. the reference human genome (USCS hg38) using the Burrows–Wheeler Aligner (BWA) version 0.7.10 (for Clinical Assessment more information, see github.com/lh3/bwa). All the mutations detected by sequencing were annotated The clinical criteria for the inclusion of patients using the 1000 Genomes Project (http://1000genomes. in this study included a decrease in central vision org/), dbSNP (http://www.ncbi.nlm.nih.gov/projects/ unrelated to refraction, and a different degree of SNP/), ExAC (http://exac.broadinstitute.org), optic nerve atrophy observed through fundus examina- and ESP6500 (http://evs.gs.washington.edu/EVS/) tion. We eliminated all known causes, such as oppres- databases, screening for mutations with minor allele sion, inflammation, glaucoma, and drug-induced optic frequency <0.1% to detect possible deleterious varia- nerve atrophy. All participants in the study underwent tions. In addition, four online tools, Sorting Intolerant complete ophthalmologic examinations, comprising from Tolerant (http://sift.jcvi.org/), MutationTaster

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 4

Figure 1. Basic information of clinical presentation and genetic finding of the ntDNA in the patients. (A) The age distribution ofthetotal patients, including age ≤10 years (n = 99), 10 to 20 years (n = 86), 20 to 30 years (n = 78), 30 to 40 years (n = 67), and >40 years (n = 88). (B) The patients received a confirmed genetics diagnosis in this cohort, including mtDNA (n = 132), OPA1 (n = 36), OPA3 (n = 4), and homozygous TMEM126A (n = 2) variants. (C) Thirty-eight different pathogenic/likely pathogenic nuclear variants were detected in this cohort, including OPA1 gene (n = 30), TMEM126A gene (n = 4), RTN4IP1 gene (n = 3), and OPA3 gene (n = 1). (D) Thirty different OPA1 pathogenic/likely pathogenic variants were identified, including missense (n = 11), splicing (n = 7), nonsense (n = 5), frameshift (n = 5), and intron (n = 2) variants. (E, F) Multidimensional comparison between detection of variability in patients and age distribution.

software (http://www.mutationtaster.org/), FATHMM were screened in all subjects, as well as 19 other candi- software (http://fathmm.biocompute.org.uk/), and date LHON mutations and 21 secondary LHON LRT (http://www.genetics.wustl.edu/jflab/)wereused mutations. Conservatism of mitochondrial genome to predict potential pathogenic variants. Then, the variation was obtained by analysis using ClustalX (see Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/), http://www.clustal.org/clustal2/). HGMD (http://www.hgmd.cf.ac.uk/ac/index.php), and Online Mendelian Inheritance in Man (OMIM, http://www.omim.org/) were used to interpret and Results prioritize the variants, combined with potential deleterious impacts, genotype-phenotype relationships, and Clinical Presentation and Genetic Findings variants report. According to the American College of Medical Genetics (ACMG) and genomics guidelines, In this cohort, a total of 418 patients with hered- variants were classified into five categories, including itary optic neuropathy were recruited in the study, of pathogenic, likely pathogenic, novel variants of uncer- whom most were unrelated probands (341/418). The tain clinical significance, likely benign, and benign. average age of the patients was 25.7 ± 16.5 years (range, Sanger sequencing was performed to confirm the 0.83–66; median, 23), most of whom were under candidate variants.29,30 30 years old, accounting for (63%; 263/418) of the The mtDNA genome-wide sequencing results total number of patients (Fig. 1A), and all participants were compared with the published mtDNA were from all over the Chinese region. Among these sequences (GenBank Accession No. NC_012920), patients, 263 were men and 155 were women, with an and the mtDNA haplogroup status and potential average age of 23.9 ± 15.0 (range, 0.83–62; median, pathogenic variation were both determined. The 22) years and 28.6 ± 18.3 (range, 2–66; median, 27) “Top 19” primary LHON mutations reported by years, respectively (Table 1). The average ages of the MITOMAP (https://www.mitomap.org/foswiki/bin/ two groups of dominant optic atrophy and LHON view/MITOMAP/MutationsLHON) in the literature patients were calculated, and the results showed that

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 5 Table 1. The Demography and the Genetic Screening Results of this Study Total Male Female Ratio (Male Patients (%) (Families/Sporadic) (Families/Sporadic) (Families/Sporadic) to Female) Total screened (100%) 418 (254/164) 263 (154/109) 155 (100/55) 1.7:1 LHON mutation (31.6%, 132/418) 132 (84/48) 90 (55/35) 42 (29/13) 2.1:1 OPA1 mutation (8.6%, 36/418) 36 (30/6) 22 (18/4) 14 (12/2) 1.6:1 OPA3 mutation (1.0%, 4/418) 4 (3/1) 3 (3/0) 1 (0/1) 3.0:1 TMEM126A mutation (0.5%, 2/418) 2 (2/0) 1 (1/0) 1 (1/0) 1.0:1 Total detected (40.2%, 168/418) 168 (113/55) 111 (72/39) 57 (41/16) 2.0:1

the age of onset of dominant optic atrophy in patients of male to female was 2.1:1. Molecular and clinical was younger than with LHON, with an average age of features of LHON-positive individuals identified in 19.8 ± 14.9 (range, 3–54; median, 12) years and 27.1 the study are shown in Table 2. The top 3 primary ± 15.7 (range, 1–66; median, 25) years, respectively. In LHON disease-causing mutation were detected in 96 addition, 400 unrelated control subjects were included patients, of which 84 patients detected m.11778G>A from different regions of China, with an average age mutation, 7 patients detected m.3460G>A mutation, of 48.5 ± 15.0 (range, 22–92; median, 48) years, who and 5 patients detected m.14484T>C mutation. In showed no abnormalities through physical, neurologic, addition, 11 other candidate LHON disease-causing and ophthalmic examinations. mutations were found in 52 patients, and 5 putative Overall, a total of 168 patients (40.2%; 168/418) LHON mutations were detected in 40 patients. The received confirmed mtDNA or ntDNA variants, and primary top 3 LHON mutations accounted for 72.7% the percentage of patients diagnosed with confirmed (96/132) of the patients with LHON mutations, and 16 genetic variants in the families (44.5%; 113/254) was mtDNA/mtDNA coexisting mutations accounted for significantly higher than that of sporadic patients 12.1% (16/132) (Table 3). (33.5%; 55/164) (Table 1). As shown in Figure 1B, a The m.11778G>A mutation was detected in 114 total of 132 individuals (76%; 132/174) were detected individuals, including 84 patients and 30 unaffected with LHON-mtDNA mutations, 36 individuals were family members. The affected individuals presented detected with OPA1 gene mutations, 4 individuals bilateral painless visual impairment along with were detected with OPA3 gene mutations, and 2 scotomas (central or cecocentral) and pale optic patients were detected with homozygous TMEM126A disc. Family members who harbor the primary gene mutations. We performed a multidimensional m.11778G>A mutation were considered as unaffected comparison between the type of variation and the individuals when they did not show any symptoms age distribution of the patients examined. The results of visual impairment at the time of sample collec- showed that the detection rate of mtDNA mutations tion. Among these patients who harbor the primary was almost the same in patients of different ages, m.11778G>A mutation, 56 patients were male (66.7%, but OPA1 mutations were mainly concentrated in 56/84) and 28 were female (33.3, 28/84); the ratio of children under 10 years of age (Figs. 1E, 1F). Coexist- male to female was 2:1. When compared with the ing variants were detected in 27 patients (16.1%; females, the penetrance of the disease was much higher 27/168), including 16 mtDNA/mtDNA variants in males, with 91.8% of males (56/61) who carried the (59.2%; 16/27), 4 ntDNA/ntDNA variants (14.8%, primary m.11778G>A mutation developing blindness, 4/27), and 7 mtDNA/ntDNA variants (25.9%, 7/27). whereas 52.8% of females (28/53) presented with loss Among these coexisting variations, the commonest of vision. The observation was consistence with previ- form was ND4 m.11778G>A+ND6 m.14502T>C, ous European and Chinese studies, indicating that followed by ND4 m.11778G>A+ND1 m.3394T>C, gender factors have a strong impact on the increased or ND6 m.14484T>C+ND1 m.3394T>C, all three risk of visual impairment in males compared with forms have been reported. females who harbor the m.11778G>A mutation in Chinese cohort.31,32 mtDNA LHON Mutations As shown in Table 1, LHON-mtDNA mutations OPA1 and OPA3 Mutations were detected in 132 patients (31.6%, 132/418), of which 84 were families subjects (63.6%, 84/132) and Thirty-eight distinct pathogenic/likely pathogenic 48 were sporadic subjects (36.3%, 48/132); the ratio nuclear variants were detected in this cohort

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 6 Yes Yes Report Previous (2; 28.6%) (Num; %) The Most Common M9 (2; 40%) Yes Haplogroup M7 (12; 14.3%) M7 (14; 14.6%) D4 (20; 23.8%); D4 (20; 20.8%); F1 (3; 42.9%); M7 17.5 11.0 16.0 15.9 ––Yes ––Yes ––Yes ––Yes ± ± ± ± Age of Standard (1–66;15) (8–36; 23) (12–55; 40) Deviation ± (0.83–66; 25.5) (Range; Median) Diagnosed Mean Clinical Pathogenic Pathogenic Pathogenic Pathogenic PathogenicPathogenic – – – – Yes Yes Pathogenic – – Yes Pathogenic 36.8 Pathogenic 21.6 Pathogenic 28.7 Significance damaging damaging damaging damaging damaging damaging Phenotyping Polymorphism of 400 N Controls Clinvar of 418 N Patients Index Conservation AA 0.9556 0.9333 1 1 0 Pathogenic 0 Pathogenic Probably Benign Pathogenic – – Yes A 1 84 2 Pathogenic Probably A 0.25 2 0 Pathogenic Benign Risk factors; C 0.4222 1 0 Pathogenic Benign Risk factors; C 0.83 5 0 Pathogenic Probably C 0.83 16 2 Pathogenic Unknown Risk factors; A 0.9333 2 0 Pathogenic Probably A 0.9111 7 0 Pathogenic Probably G 0.9778 2 0 Pathogenic Probably C 0.9333 8 2 Pathogenic Benign Risk factors; > > > > > > > > > > > Molecular and Clinical Features of LHON-Positive Patients Identified in this Study 11253T 10197G 10680G 3394T 3635G 4136A 3460G 14484T 11778G 11696G 12338T ND4 ND3 ND4 Total of the “top 3”primary LHON-disease causing mutationsND1 ND1 ND1 28.6 ND1 ND6 Gene Mutation Table 2. ND4 ND4 ND5

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 7 Report Previous (Num; %) The Most Common Haplogroup F2 (11; 21.2%) M10 (11; 21.2%); 14.8 ––Yes ––Yes ––Yes ± Age of Standard (3–55; 22) Deviation ± (Range; Median) Diagnosed Mean Clinical Pathogenic Pathogenic Pathogenic Risk factors; Significance damaging Phenotyping Polymorphism Benign Risk factors – – Yes Benign Risk factors – – Yes Benign Risk factors – – Yes Benign Risk factors – – Yes – Risk factors – – Yes pathogenicity significance significance pathogenicity pathogenicity of 400 N Controls Clinvar of 418 N Patients Index Conservation A 0.3333 17 17 Conflicting of G 0.78 7 0 Pathogenic Probably C 0.7778 11 1 Pathogenic Benign Risk factors; C 0.5556 16 17 Uncertain A 0.9333 1 0 Pathogenic – Risk factors; A 0.9333 2 1 Conflicting of G 0.9111 1 2 Uncertain C 0.2444 4 3 Conflicting of > > > > > > > > Continued 4216T 14502T 4917A 9438G 15951A 12811T 13708G 9804G Thr ND1 Table 2. ND6 Gene Mutation ND2 COX3 tRNA- Total of the other candidate LHON disease-causing mutations 24.5 ND5 ND5 COX3 Total of the putative LHON mutation.

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 8 Table 3. Coexistence Variation Identified in the Patients Age of Diagnosed N of 418 Mean ± Standard Deviation Mutation Patients (%) (Range; Median) Haplogroup (num; %) ND4 m.11778G>A+ND4 m.11696G>A 1 25 D4j ND4 m.11778G>A+ND6 m.14502T>C 5 30.8 ± 17.6 (9–54; 30) M10a1 ND4 m.11778G>A+tRNA-Thr 3 19.3 ± 15.5 (8–37; 13) D4h m.15951A>G ND4 m.11778G>A+ND1 m.3394T>C326± 14.8 (16–43; 19) M9a1a2 ND6 m.14484T>C+ND1 m.3394T>C 3 29.7 ± 19.1 (12–50; 27) M9a1a1 (2); M7b1a1 (1) ND1 m.4136A>G+COX3 m.9438G>A155 N9a7 Coexistence variation of 16(3.8%) 28.7 ± 16.1 (8–55; 26) M7/9/10 (11; 68.8%); D4 mtDNA/mtDNA (4; 25%); N9 (1; 6.2%) OPA1 c.1682-1G>A+OPA3 c.123C>G213± 4.2 (10–16; 13) D4a (1); B5a2a2 (1) OPA1 c.1471dup(p.Ile491Asnfs*9) 225± 19.8 (11–39; 25) B4h1 /c.1472T>A+TMEM126A c.236C>G,Het Coexistence variation of ntDNA/ntDNA 4 (1%) 19 ± 13.6 (10–39; 13.5) B (3; 75%); D4 (1; 25%) ND1 m.12338T>C+OPA1 c.2203G>T229± 26.9 (10–48; 29) F2a1 (1); H5g (1) ND1 m.12338T>C+RTN4IP1 c.308G>A, 110 F2 Het ND1 m.4136A>G+OPA1 1 4 G2a1 c.1835_1836delGA ND4 m.10680G>A+OPA1 15 A17 c.2661+1G>A ND4 m.11778G>A+TMEM126A 1 16 D5a2a c.163C>T,Het ND4 m.11253T>C+RTN4IP1 138 G1 c.1015C>T,Het Coexistence variation of mtDNA/ntDNA 7(1.7%) 18.7 ± 17.3 (4–48; 10) F2 (2; 28.6); G (2; 28.6);D (1; 14.3%); A (1; 14.3%); H (1; 14.3%) Total patients of coexistence variation 27(6.5%) 24.7 ± 16.3 (4–55; 19) Top 3 haplogroup: M10a (5; 18.5%); M9a (5; 18.5%); D4 (5; 18.5%)

(Supplementary Table S2), including OPA1 gene (n = the patient’s biological parents, we defined these as de 30, 79%), TMEM126A gene (n = 4, 10%), RTN4IP1 novo variants. In this cohort, two de novo missense gene (n = 3, 8%), and OPA3 gene (n = 1, 3%) (Fig. 1C). variants were detected in the OPA1 gene (1.43%, We identified 30 distinct pathogenic/likely pathogenic n = 2/140 families of trios), c.320C>A (p.S107X), variants of the OPA1 gene in 8.6% (36/418) of patients, and c.1499G>A (p.R500H) were detected in unrelated including missense (n = 11, 36%), splicing (n = 7, patients, respectively. 23%), nonsense (n = 5, 17%), frameshift (n = 5, 17%), One recurrent OPA3 mutation was detected in four andintron(n= 2, 7%) variants (Fig. 1D). Among (1.0%) of the 418 patients screened. Of these four the 30 OPA1 mutations, we classified 16 variants as patients, two of them were from the same family (F74), novel variants because these had not been registered and the other two were from independent families in the Human Gene Mutation Database or Clinvar (F159, F305). The variant c.123C>G (p.I41M) database and have not been reported in the literature. was reported to be detected in a 12-year-old boy In addition, when the variants were not detected in with a BCVA of 0.1. Ophthalmoscopy examination

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 9

Figure 2. Detection and evaluation of mutations in the TMEM126A gene. (A) Multiple sequence alignment of candidate missense mutations from different species to explore the conservation of these mutations. The red arrow represents variants states. (B) SWISS-MODEL was used to predict the three-dimensional structure of both the mutant and wild-type proteins. (C) Pedigrees of the families with mutations. Squares indicate men and circles women; black and white symbols represent affected and unaffected individuals, respectively. The proband is marked with an arrow,andtheasterisks indicate those members enrolled in this study.

revealed bilateral optic disc pallor and diffuse defects TMEM126A and RTN4IP1 Mutations in the retinal nerve fiber layer (RNFL). His twin broth- > ers also harbors the same mutation, as well as visual One missense mutation (c.107C T, p.S36L) of acuity defects and optic atrophy.33 Moreover, the the TMEM126A gene was identified in five members clinical significance of these variants was defined of family 98 (F98), the probands and his affected according to ACMG and genomics guidelines; biological brother harbor homozygous state, and his the bioinformatics tools analysis results of the 17 parents and sister harbor heterozygous state (Fig. 2C). missense variants are demonstrated in Supplementary This missense variant affects a highly evolutionarily Figure S1. conserved region (Fig. 2A) and changes the amino acid

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 10 from serine to leucine, and the variant segregates with variant of haplotype F (F8), which has been reported the disease within the family. The SWISS-MODEL to detect homozygous and heterozygous variation of was used to predict the three-dimensional structure of the mutation in four families.24 this amino acid in the mutant and wild-type proteins (Fig. 2B), and we observed a difference between the mutated and wild-type structures of transmembrane Haplogroup Distribution protein 126A. There have not been any other LHON- Our comprehensive study of Chinese LHON mtDNA and ADOA and AROA genes detected in patients confirmed that LHON individuals with the two affected subjects of this family. Studies have m.11778G>A variants had different mtDNA haplo- reported that the two affected siblings from Iraqi types, which is different from previously reported descent detected the p.S36L variant and catego- observations that preferentially associate with Western rized the variant as unclear significance.34 However, European continent haplogroup J and increase clinical according to the ACMG and genomics guidelines, penetrance of the J2 subhaplogroup, indicating their the p.S36L variant is cosegregated with the disease in possible influence on clinical expression.31,35 Compre- the family and affects evolutionary conserved amino hensive haplogroup analysis showed that 34.5% (29/84) acid residues. Therefore it should be categorized as and 28.6% (24/84) of the affected subjects belonged to pathogenic or likely pathogenic variants, so we regard haplogroup D and M, with a high frequency of subhap- it as a candidate causative factor for the patients with logroups D4, D5, and M7 (Supplementary Table S3). optic atrophy in this family. These data suggest that such haplotypes are more likely Moreover, we detected three additional heterozy- to produce similar clinical manifestations in East Asian gous states of the TMEM126A gene in affected populations, and the same situation may exist in other and unaffected individuals (c.163C>T, p.Arg55Ter; mitochondrial diseases among Chinese populations. c.62T>G, p.Ile21Ser; c.236C>G, p.Thr79Arg), includ- Moreover, we performed mitochondrial haplogroup ing the detection of a recurrent nonsense mutation comparison between the 400 control subjects and 418 (p.Arg55Ter) in three individuals of a family (F147).5,26 patients, the result indicating that there is no difference Among the three mutation carriers, the proband was between these two cohorts (Supplementary Table S4). accompanied by an acute onset of optic neuropathy, and the other two carriers were asymptomatic fathers and aunts. Recurrent nonsense mutations (p.Arg55Ter) detected in the proband were inherited from his Discussion father. At the same time, LHON-mtDNA mutation m.11778G>A was also detected in the proband and To our knowledge, this is the first study screen- his four family members, and the proband’s mtDNA ing for LHON, ADOA, and AROA in a large cohort mutation was inherited from his mother (Fig. 2C). of Chinese patients with suspected hereditary optic The proband showed more severe clinical manifesta- neuropathy. Comprehensive molecular screening for tions than the infected mother, and the onset age was patients with suspected hereditary optic neuropathy earlier (acute onset at the age of 16 years), and the might contribute to the high overall mutation detection vision dropped sharply in early childhood. However, rate (40.2%, 168/418). Our results indicate that LHON- the affected mother did not have a similar history of mtDNA mutations are definitely the most common the disease; she was late onset and vision decrease cause in Chinese patients, and mtDNA variants were was mild. The effect of the coexistence of mtDNA found in 31.6% (132/418) of patients. The LHON- variants (m.11778G>A) and TMEM126A heterozy- mtDNA mutation detection rate is higher than the gous variants (c.163C>T, p.Arg55Ter) on clinical incidence of OPA1 mutations, which were detected in manifestation is unknown, and its underlying mecha- less than 10% (8.6%, 36/418) of patients. However, the nism remains to be explored. detection rate of LHON-mtDNA mutations is 31.6% In three families with early-onset recessive optic (132/418), which is a discrepancy to 52.1% mutation neuropathy, we identified three heterozygous missense rate (271/520) based on another Chinese cohort.33 In states in the RTN4IP1 gene (c.1015C>T, p.R339C; additional, these finding are in sharp contrast to the c.308G>A, p.R103H; c.962G>C, p.G321A), which results of 980 unrelated patients in the large French encodes a mitochondrial ubiquinol oxydo-reductase. population, which showed that the OPA1 mutation Among these three mutations, two of them were was the commonest mutation detected in 30% of novel heterozygous mutations for the c.1015C>Tand probands, whereas the LHON-mtDNA mutation was c.962G>C substitution on a different haplotype F and only detected in 13% of patients.20 We believe that C7 (F120 and F148), another one is a heterozygous the difference in the detection rate with the French

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 11

population is due to different ethnic backgrounds, and m.14502T>C).17,38 In the current study, a total of 27 the difference in the detection rate between studies patients were found to have one of three coexisting based on the Chinese population is due to differ- variants, including 16 mtDNA/mtDNA variants (3.8%, ent entry criteria. Our enrollment included a broader 16/418; 28.7 ± 16.1), 4 ntDNA/ntDNA variants (1%, group of patients with suspected optic neuropathy, 4/418; 19 ± 13.6), and 7 mtDNA/ntDNA variants which may lead to a low detection rate, but the results (1.7%, 7/418; 18.7 ± 17.3) (Table 3). To our knowledge, show that mtDNA or OPA1 mutations are indeed this is the first report of the simultaneous detection of detected in some patients with clinical symptoms that three forms of coexisting variants detected in Chinese are not obvious. In clinical applications, a comprehen- cohort studies. In particular, the mtDNA/ntDNA sive examination helps us to diagnose patients with variant forms are very unique, and novel forms suspected optic neuropathy. The prevalence of LHON- of mutations might cause more severe impact on mtDNA and OPA1, OPA3 mutations was similar to subjects harboring the coexist variants. Among the the results of one independent large Chinese cohort seven mtDNA/ntDNA variants, one patient was found study from a domestic study group. In this large-scale harboring m.11778G>A and c.163C>T mutations, cohort study, mutations in the mtDNA were detected compared with the mother who only detected the in 346 probands (38.3%, 346/903) who were suspected m.11778G>A mutation, the probands who detected with LHON from 903 Chinese families. G11778A, m.11778G>A and c.163C>T mutations showed an T14484C, and G3460A mutations were detected in earlier acute onset. 312 (90.2%), 30, and 4 probands, respectively.36,37 However, the top three LHON-mtDNA mutations were detected in 28.2% (96/341) of the screened Conclusions probands in our cohort study, G11778A, T14484C, and G3460A mutations were detected in 84 (87.5%), 5, and 7 probands, which accounted for 72.7% (96/132) LHON-mtDNA mutations are the commonest of all identified LHON-mtDNA mutations. Consistent genetic defects in this Chinese cohort with suspected with several previous reports, OPA1 is the common- hereditary optic neuropathy, followed by the OPA1 est disease-causing gene in Chinese ADOA patients, and OPA3 mutations with ADOA, and TMEM126A OPA3 gene mutations were rare in Chinese cohort, mutations with the rare AROA. Comprehensive molec- and we only detected one missense variant c.123C>G ular screening for patients with suspected hereditary (p.I41M) in four patients in our cohort study.36 In optic neuropathy is essential to assist in diagnosing the addition to detecting of the two most common forms patient’s disease categories and customize molecular of optic neuropathy in LHON-mtDNA and ADOA, therapies. Three forms of coexisting variation may have four variants of the TMEM126A gene and three potential effects on disease manifestation and disease variants of the RTN4IP1 gene were also detected in this progression, a task that requires further exploration cohort study. and in-depth research. Except for the three commonest LHON-mtDNA mutations (11778G>A, 14484T>C, 3460G>A), 11 other candidate LHON disease-causing mutations Acknowledgments were detected in 12.4% (52/418) of patients, and more than 6.5% (27/418) of patients were detected to harbor The authors thank all of the patients and families two variants (one of three coexisting variant forms). who agreed to participate in this study. In addition, In several previous studies, the coexisting variants the authors thank BGI Shenzhen for their technical forms of mtDNA/mtDNA or mtDNA/ntDNA or support, and the staff at the Eye, Ear, Nose, and ntDNA/ntDNA detected in one patient was very rare. Throat Hospital of Fudan University for their assis- In several recent studies, six unrelated patients harbor- tance. Finally, the authors are grateful to Fang Chen, ing m.14484T>C and m.14502T>C mutations were Ji-Hong Wu, and Jie Huang for their invaluable contri- detected in a large cohort study, which included 1218 butions to this work. Han Chinese patients; in another Chinese cohort study of 520 unrelated patients, nine coexisting variants were Supported by the National Natural Science detected, including one patient harboring two common Foundation of China (Grant NSFC 61373048), primary mutations (m.3460G>A and m.11778G>A); Shenzhen Municipal Government of China (No. the remaining eight patients harbor one of the top JCYJ20170412152854656), the Non-Profit Central three LHON mutations combined with one of the two Research Institute Fund of Chinese Academy of rare primary LHON mutations (m.11696A>Gand Medical Sciences 2018PT32019, Shanghai Municipal

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 12 Science and Technology Major Projects (Grant ropathy with encephalopathy and cerebellar atro- 2018SHZDZX05), the Natural Science Foundation of phy. JMedGenet. 2014;51:834–838. Guangdong Province (Grant 2018A0303100012), and 7. Zou X, Guo X, Su H, et al. Whole exon sequencing a grant from the Research Grants Council of the Hong identifies two novel mutations in the reticulon 4– Kong Special Administrative Region, China (CityU interacting protein 1 gene in a Chinese family with 11256116 and CityU 11210119). autosomal recessive optic neuropathies. J Mol Neu- rosci. 2019;68:640–646. FC, J-HW, JH, J-KL, WL, and F-JG conceived 8. Yen MY, Wang AG, Wei YH. Leber’s hereditary and designed this study. FJG, FYH, SHZ, and JHW optic neuropathy: a multifactorial disease. Prog recruited patients, performed clinical examinations and Retin Eye Res. 2006;25:381–396. interpretation. FJG, FYH, SHZ, JHW, S-FQ, JH, L- 9. Maresca A, Morgia CL, Caporali L, et al. The optic LL, and L-SW collected the clinical samples and clini- nerve: a “mito-window” on mitochondrial neu- cal data. WL, J-KL, FC, YQ, and Z-WW analyzed rodegeneration. Mol Cell Neurosci. 2013;55:62–76. the sequencing data. J-KL, WL, and F-JG wrote and 10. Harding AE, Sweeney MG, Govan GG, et al. Pedi- revised the manuscript. gree analysis in Leber hereditary optic neuropathy families with a pathogenic mtDNA mutation. Am Data availability: The data that support the findings J Human Genet. 1995;57:77. of this study have been deposited in the CNSA (https:// 11. Wallace DC, Singh G, Lott MT, et al. Mitochon- db.cngb.org/cnsa/) of CNGBdb with accession number drial DNA mutation associated with Leber’s hered- CNP0000453. The datasets are available from the itary optic neuropathy. Science. 1988;242:1427– corresponding author on reasonable request. 1430. 12. Ruiz-Pesini E, Lott MT, Procaccio V, et al. An Disclosure: J.-K. Li, None; W. Li, None; F.-J. Gao, enhanced MITOMAP with a global mtDNA None; S.-F. Qu, None; F.-Y. Hu, None; S.-H. Zhang, mutational phylogeny. Nucleic Acids Res. None; L.-L. Li, None; Z.-W. Wang, None; Y. Qiu, 2007;35:D823–D828. None; L.-S. Wang, None; J. Huang, None; J.-H. Wu, 13. Zhang M, Zhou X, Li C, et al. Mitochondrial hap- None; F. Chen, None logroup M9a specific variant ND1 T3394C may have a modifying role in the phenotypic expression * J-KL, WL, and F-JG contributed equally as first of the LHON-associated ND4 G11778A muta- authors. tion. Mol Genet Metab. 2010;101:192–199. 14. Xie S, Zhang J, Sun J, et al. Mitochondrial hap- References logroup D4j specific variant m.11696G > a(MT- ND4) may increase the penetrance and expressivity 1. Neuhann T, Rautenstrauss B. Genetic and pheno- of the LHON-associated m.11778G > a mutation typic variability of optic neuropathies. Expert Rev in Chinese pedigrees. DNA Seq. 2017;28:434–441. Neurother. 2013;13:357–367. 15. Yu-Wai-Man P, Griffifiths PG, Burke A, et al. The 2. Delettre C., Lenaers G, Griffoin JM, et al. Nuclear prevalence and natural history of dominant optic gene OPA1, encoding a mitochondrial dynamin- atrophy due to OPA1 mutations. Ophthalmology. related protein, is mutated in dominant optic atro- 2010;117:1538–1546. phy. Nat Genet. 2000;26:207–210. 16. Lenaers G, Hamel C, Delettre C, et al. Dominant 3. Reynier P, Amati-Bonneau P, Verny C, et al. OPA3 optic atrophy. Orphanet J Rare Dis. 2012;7:46 gene mutations responsible for autosomal dom- 17. Satoh M, Hamamoto T, Seo N, Kagawa Y, Endo inant optic atrophy and cataract. JMedGenet. H. Differential sublocalization of the dynamin- 2004;41:e110. related protein OPA1 isoforms in mitochondria. 4. Carelli V, Ross-Cisneros FN, Sadun AA. Mito- Biochem Biophys Res Commun. 2003;300:482–493. chondrial dysfunction as a cause of optic neu- 18. Huizing M, Dorward H, Ly L, et al. OPA3, ropathies. Prog Retin Eye Res. 2004;23:53–89. mutated in 3-methylglutaconic aciduria type III, 5. Hanein S, Perrault I, Roche O, et al. TMEM126A, encodes two transcripts targeted primarily to mito- encoding a mitochondrial protein, is mutated in chondria. Mol Genet Metab. 2010;100:149–154. autosomal-recessive nonsyndromic optic atrophy. 19. Thiselton DL, Alexander C, Taanman JW, et al. Am J Human Genet. 2009;84:493–498. A comprehensive survey of mutations in the opa1 6. Metodiev MD, Gerber S, Hubert L, et al. Muta- gene in patients with autosomal dominant optic tions in the tricarboxylic acid cycle enzyme, aconi- atrophy. Invest Ophthalmol Vis Sci. 2002;43:1715– tase 2, cause either isolated or syndromic optic neu- 1724.

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 13 20. Ferre M, Bonneau D, Milea D, et al. Molecu- thy in Chinese families with the m.11778G→A lar screening of 980 cases of suspected hereditary mutation. Am J Human Genet. 2008;83:760– optic neuropathy with a report on 77 novel OPA1 768. mutations. Hum Mutat. 2009;30:E692–E705. 33. Chen J, Xu K, Zhang X, et al. Mutation screening 21. Ferré M, Caignard A, Milea D, et al. Improved of mitochondrial DNA as well as OPA1 and OPA3 locus-specific database for OPA1 mutations allows in a Chinese cohort with suspected hereditary optic inclusion of advanced clinical data. Human Mutat. atrophy. Invest Ophthal Vis Sci. 2014;55:6987– 2015;36:20–25. 6995. 22. Hartmann B, Wai T, Hu H, et al. Homozy- 34. Kloth K, Synofzik M, Kernstock C, et al. Novel gous YME1L1mutation causes mitochondriopa- likely pathogenic variants in TMEM126A identi- thy with optic atrophy and mitochondrial network fied in non-syndromic autosomal recessive optic fragmentation. Elife. 2016;5:e16078. atrophy: two case reports. BMC Med Genet. 23. Charif M, Nasca A, Thompson K, et al. Neu- 2019;20:62. rologic phenotypes associated with mutations in 35. Torroni A, Petrozzi M, D’Urbano L, et al. Hap- RTN4IP1 (OPA10) in children and young adults. lotype and phylogenetic analyses suggest that one JAMA Neurol. 2018;75:105–113. European-specific mtDNA background plays a 24. Claire A, Pierre-Olivier G, Yasmina TA, et al. role in the expression of Leber hereditary optic Recessive mutations in RTN4IP1 cause isolated neuropathy by increasing the penetrance of the pri- and syndromic optic neuropathies. Am J Human mary mutations 11778 and 14484. Am J Human Genet. 2015;97:754–760. Genet. 1997;60:1107. 25. Yu-Wai-Man P, Votruba M, Burté F, et al. A neu- 36. Yu-Wai-Man P, Shankar S P, Biousse V, et al. rodegenerative perspective on mitochondrial optic Genetic screening for OPA1 and OPA3 mutations neuropathies. Acta Neuropathol. 2016;132:789– in patients with suspected inherited optic neu- 806. ropathies. Ophthalmology. 2011;118:558–563. 26. Meyer E, Michaelides M, Tee LJ, et al. Nonsense 37. Jia X, Li S, Xiao X, et al. Molecular epidemiology mutation in TMEM126A causing autosomal reces- of mtDNA mutations in 903 Chinese families sus- sive optic atrophy and auditory neuropathy. Mol pected with Leber hereditary optic neuropathy. J Vis. 2010;16:650–664. Human Genet. 2006;51:851–856. 27. Gao FJ, Li JK, Chen H, et al. Genetic and clini- 38. Liang M, Jiang P, Li F, et al. Frequency and cal findings in a large cohort of Chinese patients spectrum of mitochondrial ND6 mutations in with suspected retinitis pigmentosa. Ophthalmol- 1218 Han Chinese subjects with Leber’s heredi- ogy. 2019;126:1549–1556. tary optic neuropathy. Invest Ophthalmol Vis Sci. 28. Li W, Wang Z, Sun Y, et al. A start codon 2014;55:1321–1331. mutation of the TSPAN12 gene in Chinese fam- 39. Almind GJ, Ek J, Rosenberg T, et al. Domi- ilies causes clinical heterogeneous familial exuda- nant optic atrophy in Denmark–report of 15 novel tive vitreoretinopathy. Mol Genet Genomic Med. mutations in OPA1, using a strategy with a detec- 2019;7:e00948. tion rate of 90%. BMC Med Genet. 2012;13: 29. Johnston JJ, Biesecker LG. Databases of genomic 65. variation and phenotypes: existing resources and 40. Wang Y, Xu M, Liu X , et al. Targeted next- future needs. Human Mol Genet. 2013;22:R27– generation sequencing extends the mutational R31. spectrums for OPA1 mutations in Chinese families 30. Stenson PD, Ball EV, Mort M, et al. The with optic atrophy. Mol Vis. 2019;25:912–920. Human Gene Mutation Database (HGMD) and 41. Chen J, Riazifar H, Guan MX, et al. Modeling its exploitation in the fields of personalized autosomal dominant optic atrophy using induced genomics and molecular evolution. Curr Protoc pluripotent stem cells and identifying potential Bioinformatics. 2012;Chapter 1:Unit1.13. therapeutic targets. Mitochondrion. 2015;24:S30. 31. Hudson G, Carelli V, Spruijt L, et al. Clinical 42. Cohn AC, Toomes C, Potter C, et al. Autosomal expression of Leber hereditary optic neuropathy dominant optic atrophy: penetrance and expressiv- is affected by the mitochondrial DNA–haplogroup ity in patients with OPA1 mutations. Am J Ophthal- background. Am J Human Genet. 2007;81:228– mol. 2007;143:656–662. 233. 43. Nakamura M, Lin J, Ueno S, et al. Novel muta- 32. Ji Y, Zhang AM, Jia Xi , et al. Mitochondrial tions in the OPA1 gene and associated clinical fea- DNA haplogroups M7b12 and M8a affect clini- tures in Japanese patients with optic atrophy. Oph- cal expression of Leber hereditary optic neuropa- thalmology. 2006;113:483–488.e1.

Downloaded from tvst.arvojournals.org on 09/26/2021 mtDNA Combined ntDNA Screening for LHON TVST | July 2020 | Vol. 9 | No. 8 | Article 11 | 14 44. Alexander C, Votruba M, Pesch UEA, et al. OPA1, 46. Liskova P, Tesarova M, Dudakova L, et al. OPA1 encoding a dynamin-related GTPase, is mutated in analysis in an international series of probands autosomal dominant optic atrophy linked to chro- with bilateral optic atrophy. Acta Ophthalmol. mosome 3q28. Nat Genet. 2000;26:211–215. 2017;95:363–369. 45. Li H, Jones EM, Li H, et al. Clinical and genetic 47. Stewart JD, Hudson G, Yu-Wai-Man P, et al. features of eight Chinese autosomal-dominant OPA1 in multiple mitochondrial DNA deletion optic atrophy pedigrees with six novel OPA1 disorders. Neurology. 2008;71:1829–1831. pathogenic variants. Ophthalmic Genet. 2018;39: 569–576.

Downloaded from tvst.arvojournals.org on 09/26/2021